Uncoupling protein 2 and aldolase B impact insulin release by modulating mitochondrial function and Ca2+ release from the ER

Summary Uncoupling protein 2 (UCP2), a mitochondrial protein, is known to be upregulated in pancreatic islets of patients with type 2 diabetes (T2DM); however, the pathological significance of this increase in UCP2 expression is unclear. In this study, we highlight the molecular link between the increase in UCP2 expression in β-cells and β-cell failure by using genetically engineered mice and human islets. β-cell-specific UCP2-overexpressing transgenic mice (βUCP2Tg) exhibited glucose intolerance and a reduction in insulin secretion. Decreased mitochondrial function and increased aldolase B (AldB) expression through oxidative-stress-mediated pathway were observed in βUCP2Tg islets. AldB, a glycolytic enzyme, was associated with reduced insulin secretion via mitochondrial dysfunction and impaired calcium release from the endoplasmic reticulum (ER). Taken together, our findings provide a new mechanism of β-cell dysfunction by UCP2 and AldB. Targeting the UCP2/AldB axis is a promising approach for the recovery of β-cell function.


INTRODUCTION
Type 2 diabetes mellitus (T2DM) occurs as a consequence of b-cell dysfunction in a background of systemic insulin resistance (Kahn, 2000). Insulin secretion from pancreatic b-cells is tightly regulated to maintain blood glucose levels within a narrow range. Mitochondrial oxidative phosphorylation (OXPHOS) is the predominant source of ATP in b-cells, and patients with mtDNA mutations exhibit b-cell dysfunction and overt diabetes due to reduced ATP production (Ereci nska et al., 1992;Gerbitz et al., 1996).
Uncoupling proteins (UCPs) create proton leaks in the mitochondrial inner membrane, which dissipate the proton motive force, thereby bypassing energy conservation by ATP synthase, and the energy is dissipated as heat. UCP1 is specifically expressed in brown adipocytes and is known to play a role in thermogenesis (Nedergaard et al., 2001). Moreover, UCP2, which has 59% amino acid identity to UCP1 in humans, is expressed widely in metabolic tissues, including pancreatic b-cells (Fleury et al., 1997). UCP2 protein levels have been reported to be increased in the islets of individuals with T2DM compared with those from nondiabetic humans (Anello et al., 2005). It remains unclear how UCP2 affects the development of diabetes, yet some research groups have used either UCP2-overexpressing mouse islets or b-cell-specific UCP2knockout mice (Chan et al., 1999;Produit-Zengaffinen et al., 2007;Robson-Doucette et al., 2011;Zhang et al., 2001). The possibility that UCP2 does not act as an uncoupler has also been suggested (Galetti et al., 2009;Nicholls, 2021).
To clarify the significance of UCP2 expression in pancreatic b-cells in relation to the pathogenesis of T2DM, we investigated the regulatory mechanisms underlying UCP2 expression in b-cells and analyzed the phenotype of mice with a b-cell-specific UCP2-overexpressing transgene (bUCP2Tg). We also focused on the role of aldolase B (AldB) in b-cells, which was the most upregulated gene in islets from bUCP2Tg mice. Figure 2. bUCP2Tg mice showed impaired glucose tolerance with reduced insulin secretion (A) Ucp2 mRNA levels in the islets, livers, and hypothalamus from bUCP2Tg mice and their littermate WT mice (WT: n = 4, bUCP2Tg: n = 5-6). (B) Blood glucose levels in bUCP2Tg and WT mice (WT: n = 13, bUCP2Tg: n = 21). (C) OGTT in bUCP2Tg and WT mice (WT: n = 29, bUCP2Tg n = 36, 1.5 g/kg BW glucose). (D) Body weight changes in bUCP2Tg and WT mice (WT: n = 13, bUCP2Tg: n = 21). (E) Western blot of UCP2 and a-tubulin in bUCP2Tg and WT islets. Islets were incubated with 11.1 mmol/L glucose for 24 h (n = 3 per group). (F) Serum insulin levels during the OGTT in bUCP2Tg and WT mice at 0 and 15 min (left panel, WT: n = 7, bUCP2Tg: n = 8, 1.5 g/kg BW glucose) and at 0, 2, and 5 min (right panel, WT: n = 14, bUCP2Tg: n = 17, 2.5 g/kg BW glucose). (G) Glucose-stimulated insulin secretion (GSIS) in bUCP2Tg and WT islets. Islets were incubated in KRB buffer containing 2.8 or 16.7 mmol/L glucose for 90 min with or without 100 nmol/L liraglutide (n = 5 per group). (H) Western blot of UCP2 and a-tubulin in MIN6-M9 cells. Cells were infected with Ad-LacZ or Ad-Ucp2 at an MOI of 500 for 48 h (n = 3 per group). (I) GSIS in MIN6-M9 cells. Cells were infected with Ad-LacZ or Ad-Ucp2 at an MOI of 500 for 48 h and then incubated with 2.8 or 16.7 mmol/L glucose for 90 min (n = 4 per group).

OPEN ACCESS
iScience 25, 104603, July 15, 2022 3 iScience Article evaluate the effect of glucose signaling on changes in Ucp2 expression, we treated the islets with glucose, a glucokinase activator (GKA) ( Figure 1E), or insulin ( Figure 1F), and in all conditions, the expression of Ucp2 was increased. Moreover, the upregulation of Ucp2 induced by high glucose concentration was partially inhibited by N-acetylcysteine (NAC), an antioxidant ( Figure 1G). We also confirmed the increased protein levels of UCP2 in mouse islets and INS1 b-cells in the presence of high glucose concentrations (Figures 1H and 1I).
Because the activation of glucose signaling by GKA inhibited the apoptosis induced by ER stress , we examined the effect of ER stress on Ucp2 expression in mouse islets. The expression of Ucp2 in mouse islets was not changed by treatment with thapsigargin, an inducer of ER stress (Figure S1A). We next assessed the effects of insulin receptor signaling on Ucp2 expression. OSI-906, a dual inhibitor of IGF-1 and insulin receptors, had no effect on Ucp2 expression in mouse islets ( Figure S1B). Conversely, islet Ucp2 expression was decreased by treatment with Akt inhibitor X, a selective inhibitor of Akt phosphorylation ( Figure S1C), or U0126, an ERK inhibitor ( Figure S1D). We also explored the effect of potassium channel activity and calcium signaling on Ucp2 expression, which are important in glucosestimulated insulin secretion, and no changes in Ucp2 expression were observed in mouse islets treated with diazoxide, a potassium channel opener ( Figure S1E); nifedipine, an L-type calcium channel blocker; or FK506, a calcineurin inhibitor ( Figure S1F). The mammalian target of rapamycin (mTOR) signal is activated by insulin signaling in pancreatic b-cells, but the mTOR inhibitor rapamycin had no effect on islet Ucp2 expression under high glucose conditions ( Figure S1G).
These results indicated that the expression of Ucp2 was upregulated via Akt or ERK pathways under diabetes-like pathophysiological conditions, such as hyperglycemia and oxidative stress.

Overexpression of UCP2 in b-cells impairs insulin secretion
To investigate the impact of UCP2 upregulation in diabetic b-cells, we generated bUCP2Tg mice. These mice overexpress the full-length Ucp2 gene specifically in pancreatic b-cells under rat insulin promoter activity ( Figure S2A). bUCP2Tg mice showed significantly increased Ucp2 expression in islets but not in livers or hypothalamus compared with the corresponding wild-type (WT) mice ( Figure 2A). bUCP2Tg mice exhibited significantly impaired glucose tolerance (Figures 2B and 2C). No changes were observed in body weights in bUCP2Tg mice compared with the controls ( Figure 2D). We then validated the increased protein levels of UCP2 in islets from bUCP2Tg mice (bUCP2Tg islets) ( Figure 2E). Moreover, there was no change in the expression level of Ucp1 in bUCP2Tg islets ( Figure S2B), suggesting an absence of compensatory effect between the isoforms. Serum insulin levels were significantly reduced in bUCP2Tg mice at 5 but not 15 min after glucose loading ( Figure 2F). The insulinogenic index was decreased in bUCP2Tg mice at 2 and 5 min after glucose loading ( Figure S2C). The bUCP2Tg islets showed decreased insulin secretion in response to glucose stimulation ( Figure 2G). Conversely, liraglutide, a GLP-1 receptor agonist, enhanced insulin secretion in both mice ( Figure 2G). Furthermore, adenovirus-vector-induced expression of Ucp2 reduced insulin secretion in MIN6-M9 cells and human islets ( Figures 2H-2J and S2D). The insulin sensitivity in bUCP2Tg mice was similar to that in WT mice ( Figure 2K). b-cell mass and b-cell proliferative activity in bUCP2Tg mice were comparable to those in WT animals ( Figures S2E and S2F).
bUCP2Tg islets exhibited abnormalities in mitochondrial morphology and function Because UCP2 localizes to the inner mitochondrial membrane, we investigated mitochondrial function in bUCP2Tg islets. The bUCP2Tg islets showed a significant decrease in ATP production in response to glucose stimulation ( Figure 3A). Analysis of mitochondrial respiration revealed that mitochondrial basal respiration, maximal respiration, and ATP production were all significantly lower, whereas no change in proton leakage was noted in bUCP2Tg islets ( Figures 3B and 3C). Furthermore, adenovirus-vector-induced overexpression of Ucp2 reduced mitochondrial membrane potential in MIN6-M9 cells, suggesting that mitochondrial dysfunction was induced by UCP2 ( Figure 3D). We found that the protein level of Figure 2. Continued (J) GSIS in human islets. Islets were infected with Ad-LacZ or Ad-Ucp2 at 3 3 10 6 MOI and cultured at 5.6 mmol/L glucose for 48 h. Then, islets were incubated in KRB buffer containing 2.8 or 16.7 mmol/L glucose for 60 min (n = 5 per group). Insulin concentration was normalized by insulin content in islets. The presented data are the ratio to the value from islets infected with Ad-LacZ control for each donor.
(K) Fold-changes in blood glucose concentrations in bUCP2Tg and WT mice in the insulin tolerance test (ITT) (WT: n = 15, bUCP2Tg: n = 12, 0.75 U/kg BW insulin). Data are the means G SEM *p < 0.05, **p < 0.01. N.S.: not significant. The two-tailed Student's t test was used in (A-F), (H), and (K). One-way ANOVA was used in (G), (I), and (J). iScience Article NDUFB8, a component of mitochondrial OXPHOS complex 1, was also significantly decreased in bUCP2Tg islets ( Figure 3E). Meanwhile, there were no significant differences in the mRNA expression of Ndufb8, a complex 1 gene, and Crif1, which is associated with the synthesis of mtDNA-encoded OXPHOS polypeptides between bUCP2Tg and WT islets ( Figure 3F). Dilatation of mitochondria with loss of cristae was observed in bUCP2Tg b-cells by electron microscopy ( Figure 3G).

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Mitophagy, which refers to selective mitochondrial autophagy, is critical for the removal of damaged mitochondria (Lemasters et al., 1998). In bUCP2Tg islets, the protein expression levels of PARKIN and PINK1, which are known to be mitophagy-related proteins (Narendra et al., 2008), were significantly decreased ( Figure 3H). It has been reported that upregulated mitophagy flux reduces the expression of PARKIN due to increased protein turnover in the MIN6 b-cell line (Sidarala et al., 2020). We measured the mitophagy flux in UCP2-overexpressing MIN6-M9 cells, and the mitophagy flux was significantly increased by UCP2 ( Figures 3I and 3J). These results indicated that the mitochondrial dysfunction induced by UCP2 enhanced mitophagy, resulting in decreased mitophagy-related protein expression. Moreover, the expression levels of Drp1, Opa1, and Mfn1, which are related to mitochondrial fission and fusion (Zorzano et al., 2010), and Tfam, a mitochondrial transcription factor (Ekstrand et al., 2004), remained unchanged in bUCP2Tg islets ( Figure 3K).

Aldolase B is induced by UCP2 in pancreatic b-cells
To identify the target genes of UCP2 in pancreatic b-cells, we performed gene expression microarray analysis in bUCP2Tg islets. We identified 13 upregulated genes (>1.5-fold, p < 0.05) and one downregulated gene (<0.67-fold, p < 0.05) in bUCP2Tg islets and validated the mRNA levels by qPCR ( Figures 4A-4C and S3A). The most upregulated gene in bUCP2Tg islets was aldolase B (AldB) ( Figures 4C and 4D), the expression of which has been reported to be negatively associated with insulin secretion in human islets (Gerst et al., 2018). The protein levels of AldB were also increased in bUCP2Tg islets ( Figure 4E). Among the aldolase subtypes, the expression of aldolase A (AldA) was the highest in the WT islets, whereas the bUCP2Tg islets showed higher expression levels of AldB than AldA ( Figure S3B). Immunofluorescence for both UCP2 and AldB was mainly localized in b-cells, and the signal intensities for these proteins were increased in bUCP2Tg islets ( Figures 4F and 4G). Stimulated emission depletion (STED) microscopy also showed increased expression of UCP2 and AldB in bUCP2Tg b-cells ( Figure S3C). We confirmed that adenovirus-vector-induced expression of Ucp2 in mouse islets increased AldB expression ( Figure 4H). The expression of UCP2 and AldB was also detected in human b-cells ( Figure S3D).
AldB expression was regulated by high glucose and free fatty acids in b-cells Next, we investigated the regulatory mechanisms of AldB expression in b-cells. The protein level of AldB in INS1 b-cells was increased by stimulation with glucose ( Figure 5A) or palmitate ( Figure 5B). Fluorescent immunostaining showed that the signal intensities for AldB were increased in human b-cells by high glucose concentrations ( Figures 5C and 5D). Consistently, the islets from hyperglycemic IRS2KO mice ( Figure 5E) and db/db mice ( Figure 5F) showed a tendency toward increased AldB expression, similar to the case iScience Article for Ucp2 expression. Treatment with insulin, however, did not affect the expression of AldB in mouse islets ( Figure S4A). Modulation of ER stress by treatment with thapsigargin ( Figure 5G) increased the expression of AldB in mouse islets, unlike the case for UCP2, suggesting that AldB expression could also be induced by cellular stressors. As shown in a previous report (Ma et al., 2007), diazoxide increased AldB expression in mouse islets ( Figure S4B). Meanwhile, neither inhibition of L-type calcium channels by nifedipine nor inhibition of calcineurin by FK-506 affected AldB expression ( Figure S4C). Regarding insulin receptor signaling, treatment with OSI-906, an IR/IGF1R dual inhibitor, decreased AldB expression, whereas treatment with an Akt inhibitor increased AldB expression in the presence of GKA ( Figures S4D and S4E). In vitro, OSI-906 did not inhibit insulin signaling in b-cells (Shirakawa et al., 2014), which could explain the difference in AldB expression between OSI-906 and Akt inhibitor.

UCP2-mediated proton leakage did not affect insulin secretion in bUCP2Tg islets
Genipin reportedly inhibited UCP2-mediated proton leakage in mouse islets (Zhang et al., 2006). The expression of AldB was not suppressed by genipin in bUCP2Tg islets ( Figure 5H). Therefore, we evaluated the contribution of proton leakage induced by UCP2 to the impairment of insulin secretion in bUCP2Tg b-cells by using genipin. Because genipin did not affect GSIS in either WT or bUCP2Tg islets ( Figure 5I), uncoupling by UCP2 did not seem to be associated with decreased insulin secretion in bUCP2Tg islets. We further investigated the effect of genipin on mitochondrial function ( Figures 5J-5L). Mitochondrial respiration after treatment with glucose or FCCP and ATP production were decreased by adenoviral expression of Ucp2 in mouse islets ( Figures 5J-5L). Proton leakage was not changed by adenoviral overexpression of Ucp2 in mouse islets ( Figure 5L), similar to bUCP2Tg islets ( Figure 3C). Moreover, genipin did not affect mitochondrial respiration, proton leakage, or ATP production in UCP2-overexpressing islets ( Figures 5K and 5L). From these results, UCP2 induced b-cell dysfunction without any changes of proton leakage.

UCP2 regulated AldB expression through the oxidative-stress-mediated pathway
We further investigated the regulatory mechanism of AldB expression by UCP2. Previous reports have shown that AldB is the target gene of hepatocyte nuclear factor 4a (HNF4a) in INS1 rat b-cell lines . We analyzed the 2-kb sequences of the murine and human AldB promoter regions and identified a putative HNF4a binding site ( Figure 6A). We also found that the expression of Hnf4a was upregulated in bUCP2Tg islets ( Figure 6B) and in WT islets exposed to high glucose ( Figure 6C). The expression of Nrf2 and Sod2, which are oxidative-stress-related genes, was increased in bUCP2Tg islets ( Figure 6D). The signal intensity of mtSOX Deep Red, which emits fluorescence in response to mitochondrial superoxide, was increased by overexpression of UCP2 in MIN6-M9 cells ( Figure 6E). We treated UCP2-overexpressing islets with NAC to evaluate the involvement of oxidative stress, and increased expression of NRF2 and SOD2 were abolished by NAC ( Figure 6F). Although the protein levels of HNF4a were not significantly changed by overexpression of UCP2 in islets, the fluorescent intensity of HNF4a in the nucleus was increased by highly expressed UCP2 in MIN6-M9 cells, and this effect was reversed by NAC treatment ( Figure 6G). We also investigated whether ER stress contributed to AldB induction by UCP2 because thapsigargin increased the expression of AldB in mouse islets ( Figure 5G). As shown in Figure S1A, Ucp2 expression was not induced by thapsigargin, and the expression of Ddit3, Atf4, Atf6, and Ern1, which are ER-stress-related genes, was not changed in bUCP2Tg islets ( Figure 6H). Furthermore, adenoviral expression of AldB in islets did not affect the expression levels of ER-stress-related genes (Figure 6I). Based on these results, oxidative stress, but not ER stress, was involved in the increase in AldB expression in bUCP2Tg b-cells.
(E) Western blot of AldB and a-tubulin in bUCP2Tg and WT islets. Islets were incubated with 11.1 mmol/L glucose for 24 h (n = 3 per group).
(F and G) Immunostaining of bUCP2Tg and WT islets. Insulin is stained red, UCP2 is stained blue, and AldB is stained green. The scale bar represents 50 mm.
(G) The intensity of insulin, UCP2, and AldB staining was calculated using ImageJ software (n = 3 per group). The intensity in the overlapping region of UCP2 and AldB staining is also represented as a merge. iScience Article expression is involved in the impaired insulin secretion in bUCP2Tg mice, we conducted AldB-knockdown experiments by using an adenovirus vector that expressed sh-RNA for AldB. Sh-AldB reduced the AldB mRNA levels in bUCP2Tg islets by approximately 50% ( Figure 6L). The knockdown of AldB restored GSIS in bUCP2Tg islets ( Figure 6M); therefore, this result indicated that AldB expression was crucial in impaired insulin secretion in bUCP2Tg mice.
Collectively, in stressed b-cells under diabetes, mitochondrial dysfunction caused by elevated UCP2 induced oxidative stress, resulting in activation of HNF4a and thus leading to the elevation of AldB expression. AldB in turn was involved in the impaired insulin secretion caused by UCP2 upregulation.

AldB expression impaired mitochondrial function in b-cells
Next, we investigated the involvement of AldB in the mitochondrial dysfunction observed in bUCP2Tg islets. Forced expression of AldB, as well as Ucp2, in MIN6-M9 cells reduced the mitochondrial membrane potential ( Figure 7A). Overexpression of AldB in mouse islets decreased the expression of mitochondrial OXPHOS proteins, complex 2 and complex 4, PARKIN and VDAC ( Figures 7B and 7C). We also confirmed that adenovirus-vector-induced AldB expression in islets had no effect on Ucp2 expression ( Figure 7D). Methylglyoxal, a major precursor of advanced glycation end products, was reportedly induced by overexpression of AldB and involved in mitochondrial dysfunction in pancreatic b-cells (Bo et al., 2016;Liu et al., 2012). We found increased levels of methylglyoxal in bUCP2Tg islets ( Figure 7E). Importantly, knockdown of AldB ameliorated the impairment of mitochondrial respiration induced by overexpression of UCP2 in islets ( Figures 7F and 7G). These results indicated that mitochondrial dysfunction in bUCP2Tg islets was mediated, at least in part, by altered AldB expression.

UCP2 and AldB reduce insulin secretion through dysregulation of Ca 2+ release from the ER in b-cells
Li et al. reported that the aldolase enzyme inhibits a TRPV4 channel to restrict Ca 2+ release from the ER under glucose starvation conditions in mouse embryonic fibroblasts (Li et al., 2019). Therefore, we investigated the roles of UCP2 and AldB in Ca 2+ release from the ER in b-cells. We confirmed the expression of Trpv2 and Trpv4 in mouse islets ( Figure 8A). HC 067047, a selective TRPV4 inhibitor, decreased GSIS in WT islets but not in bUCP2Tg islets ( Figure 8B). Fasiglifam (TAK-875), a GPR40 agonist, has been shown to enhance insulin secretion from b-cells through inositol trisphosphate (IP3)-mediated Ca 2+ release from the ER (Nolan et al., 2006). In our study, fasiglifam-induced enhancement of insulin secretion was attenuated in bUCP2Tg islets ( Figure 8C). These results suggested that UCP2 participated in the regulation of Ca 2+ release from the ER in pancreatic b-cells.
We assessed intracellular Ca 2+ influx in bUCP2Tg islets. The increase in intracellular Ca 2+ by glucose was attenuated in bUCP2Tg islets compared with islets from WT mice ( Figures 8D-8F). The fasiglifaminduced increase in intracellular Ca 2+ , after depletion of extracellular Ca 2+ levels, was completely blunted in bUCP2Tg islets, indicating that Ca 2+ release from the ER by fasiglifam was attenuated in bUCP2Tg islets ( Figures 8G and 8H). Furthermore, adenovirus-vector-induced expression of AldB in mouse islets also resulted in impaired Ca 2+ influx in response to glucose or fasiglifam ( Figures 8I-8L). These results indicated that overexpression of UCP2 and the consequent induction of AldB expression in mouse islets iScience Article resulted in impaired Ca 2+ release from the ER and reduced insulin secretion from pancreatic islets. The knockdown of AldB ameliorated the dysregulation of glucose-stimulated intracellular Ca 2+ influx caused by UCP2 overexpression in INS1 b-cell lines ( Figures 8M and 8N). We further evaluated the Ca 2+ concentration in the ER using the FRET-based probe D1ER cameleon . The ER Ca 2+ concentration in control INS1 cells slightly increased after high glucose exposure, whereas UCP2-overexpressing INS1 cells exhibited decreased ER Ca 2+ (Figures 8O and 8P). We also found that the reduction in Ca 2+ concentration in the ER after fasiglifam stimulation was not recognized by overexpression of UCP2 in INS1 b-cell lines ( Figures 8O and 8P). Although the expression levels of the Ca 2+ -binding ER protein Calr were decreased in bUCP2Tg islets, there were no changes in the mRNA expression of the Ca 2+ -ATPase pump Atp2a2, which encodes the SERCA2 protein involved in ER Ca 2+ influx or the ER Ca 2+ efflux channel Itpr1 ( Figure 8Q). The protein levels of SERCA2 also showed no changes in bUCP2Tg islets ( Figure 8R). Altogether, UCP2-mediated AldB expression might induce the dysregulation of Ca 2+ release from the ER in pancreatic b-cells. (B and C) Western blots of mitochondrial OXPHOS proteins and a-tubulin (B, n = 3 per group) and mitophagy-associated proteins and a-tubulin (C, n = 3 per group) in islets from C57BL/6J mice. Islets were infected with Ad-LacZ or Ad-AldB at 3 3 10 6 MOI in the presence of 5.6 mmol/L glucose for 48 h. (D) Ucp2 mRNA levels in islets from C57BL/6J mice. Islets were infected with Ad-LacZ or Ad-AldB at an MOI of 3 3 10 6 in the presence of 5.6 mmol/L glucose for 48 h (n = 3 per group).
(G) The graph shows nonmitochondrial oxygen consumption, basal respiration, maximal respiration, proton leakage, ATP production, and spare respiratory capacity. Data are the means G SEM *p < 0.05, **p < 0.01. N.S.: not significant. The two-tailed Student's t test was used in (A-E). One-way ANOVA was used in (F) and (G). iScience Article

DISCUSSION
In this study, we report that an increase in UCP2 expression in pancreatic b-cells induced AldB expression, which in turn impaired insulin secretion by promoting mitochondrial dysfunction and impaired Ca 2+ release from the ER ( Figure 8S).
The induction of UCP2 by high glucose or oxidative stress is consistent with several independent reports that used INS-1 b-cells, rat islets, and human islets (Brown et al., 2002;Krauss et al., 2003;Li et al., 2009;Medvedev et al., 2002;Pi et al., 2009). In the present study, we report that Akt and ERK, which are activated by glucose, are also involved in UCP2 expression in b-cells. Meanwhile, UCP2 has been shown to be upregulated by glucotoxicity in GLUTag cells, an intestinal L-cell model, and to reduce Glp-1 secretion from GLUTag cells, suggesting that UCP2 contributes to the pathogenesis of T2DM not only in b-cells (Urbano et al., 2016). Further studies are required to clarify the factors that determine UCP2 expression during the development of diabetes.
The impact of UCP2 overexpression in islets on insulin secretion in vitro remains controversial (Chan et al., 1999;Produit-Zengaffinen et al., 2007). In the current study, bUCP2Tg mice exhibited reduced insulin secretion early (5 min) after glucose loading but not at later time points (e.g., 15 min). Thus, UCP2 might be important in the early secretory response to glucose. The results of Ca 2+ imaging also showed that early elevation of intracellular Ca 2+ influx after glucose stimulation (e.g., 20 s) was attenuated in bUCP2Tg islets. We also explored the effect of the GLP-1 agonist liraglutide on GSIS in bUCP2Tg islets. GLP-1R signaling increases intracellular cAMP levels, which activate protein kinase A (PKA) and Epac2, resulting in enhanced insulin secretion in b-cells (Holst, 2007). Our data showed that liraglutide increased insulin secretion in bUCP2Tg islets as well as WT islets. Therefore, it was considered that the enhancement of insulin secretion through the GLP-1R pathway was not diminished in bUCP2Tg islets.
Dilatation of the mitochondria and decreased expression of OXPHOS proteins in the mitochondria were observed in bUCP2Tg b-cells in association with reduced ATP production. Because mitochondrial respiration was impaired without any change in proton leakage in bUCP2Tg islets, the deterioration of the electron transport chain may affect the result rather than the uncoupling. It has been shown that mitochondrial complex protein was reduced in b-cells from diabetic mice (Brown et al., 2021;Haythorne et al., 2019). Haythorne et al. also demonstrated that the protein levels of NDUFB8 were decreased in islets from diabetic iScience Article bV59M mice, but its mRNA levels were not changed, thus the downregulation of NDUFB8 protein may occur at the posttranslational level under diabetic conditions. These reports were consistent with our data, where NDUFB8 protein levels were decreased with no changes in mRNA levels in bUCP2Tg islets.
Dilatation of the mitochondria in b-cells has also been observed in diabetic db/db mice (Lo et al., 2010).
Although the protein level of PARKIN, an E3 ubiquitin ligase that plays a crucial role in mitophagy, was decreased in bUCP2Tg islets, mitophagy flux was increased. The deletion of PARKIN in pancreatic b-cells did not affect insulin secretion (Corsa et al., 2019). From these findings, it was considered that altered mitophagy-related protein levels in bUCP2Tg islets did not contribute to impaired insulin secretion and that decreased PARKIN expression was caused by the adaptive response of mitophagy and the consumption of PARKIN proteins.
We focused on the role of AldB in b-cells because its expression level was markedly upregulated in bUCP2Tg islets. Notably, the expression of both UCP2 and AldB has been reported to be increased in the islets of T2DM donors (Anello et al., 2005;Gerst et al., 2018). The expression of AldB in human islets has been reported to be negatively associated with insulin secretion in subjects with T2DM (Gerst et al., 2018). Indeed, the expression of AldB was increased in islets from diabetic db/db and IRS2KO mice, and overexpression of AldB attenuated insulin secretion in this study. We also showed an increase in the expression of AldB in human islets in response to high glucose.
We showed that oxidative stress and HNF4a might be involved in AldB transcription. HNF4a is a nuclear receptor (Nammo et al., 2008), and its binding to DNA is inhibited by AICAR, an activator of AMPK (Hong et al., 2003). The expression of downstream genes of HNF4a was suppressed by reduced nuclear localization of HNF4a in human hepatoma cells (Chandra et al., 2011). Therefore, in b-cells from bUCP2Tg mice, inactivation of AMPK by oxidative stress possibly increased the DNA binding of HNF4a and the nuclear translocation of HNF4a, which resulted in upregulation of AldB transcription. Dephosphorylation of AMPK is reportedly induced by oxidative stress in C2C12 mouse myoblast cell lines (Jiang et al., 2021). The intensity of mtSOX Deep Red, which is an indicator of mitochondrial oxidative stress, and NRF2 and SOD2 expression were increased by adenoviral expression of Ucp2 in MIN6-M9 cells or islets. Thus, the excessive UCP2 induced by glucose-induced oxidative stress might further potentiate oxidative stress that leads to AldB expression. A previous report showed the protective effect of UCP2 against oxidative stress in b-cells (Li et al., 2017). However, they also showed that superoxide levels at both 11 mmol/L and 30 mmol/ L glucose tended to be increased in UCP2-overexpressing islets. Their data indicate that oxidative stress is induced by UCP2 and are consistent with our data demonstrating the upregulation of NRF2 and SOD2 in bUCP2Tg islets.
Although it was reported that activation of NRF2 promoted mitochondrial biogenesis and insulin secretion in b-cells (Kumar et al., 2018;Yagishita et al., 2014), our UCP2 transgenic mice did not show any increase in insulin secretion. It was unclear whether the increased expression of NRF2 was involved in the phenotype of UCP2 transgenic mice, but this NRF2 upregulation was considered insufficient to restore the decreased insulin secretion in bUCP2Tg mice. Overexpression of AldB in islets reduced the mitochondrial protein levels of OXPHOS, VDAC, and PARKIN, suggesting the involvement of AldB in mitochondrial dysfunction in bUCP2Tg islets. In fact, the increase in AldB expression in MIN6-M9 cells resulted in decreased mitochondrial membrane potential. In previous reports, knockdown of AldB in endothelial cells ameliorated the high glucose-induced accumulation of methylglyoxal (Liu et al., 2012), which is known to induce mitochondrial dysfunction (Bo et al., 2016). AldB is the most upregulated protein in the islets of diabetic bV59M mice, and pathway analysis of the transcriptome data revealed a downregulation of OXPHOS in the mitochondria (Haythorne et al., 2019). Fructose 1,6-bisphosphatase 1 (FBP1), a protein that interacts with AldB, is upregulated in the b-cells of T2DM mice, and inhibition of FBP1 improves insulin secretion (Zhang et al., 2010). Taken together, AldB expression may mediate mitochondrial dysfunction, and decreased insulin secretion was observed in bUCP2Tg mice.
Our results indicated that UCP2 and AldB impaired Ca 2+ release from the ER in b-cells. Moreover, in mouse embryonic fibroblasts, aldolase binds to the TRPV4 channel under glucose starvation conditions, following activation of AMPK phosphorylation (Li et al., 2019 iScience Article b-cells. The interaction of aldolase and TRPV4 channels has been shown (Li et al., 2019). Hence, AldB might modulate the activity of TRPV4 channels. Because the intracellular Ca 2+ influx through TRPV4 channels reportedly induced IP3-mediated Ca 2+ release from the ER in endothelial cells (Heathcote et al., 2019), the interaction of TRPV4 and AldB might affect insulin secretion in bUCP2Tg b-cells through IP3-mediated Ca 2+ release. Ca 2+ release from the ER is also mediated by ryanodine receptors (RyR), and pharmacological inhibition of RyR reduces GSIS (Llanos et al., 2015). Ca 2+ release from the ER induced by GLP-1R signaling is blocked by the inhibition of RyR but not by the IP3 receptor antagonist in mouse islets and INS1 b-cell lines (Kang et al., 2001;Kim et al., 2008). Our data showed that enhancement of insulin secretion by liraglutide was preserved in bUCP2Tg islets. Therefore, it was considered that RyR-mediated Ca 2+ release from the ER was not involved in the decreased insulin secretion in bUCP2Tg b-cells.
GPR40 agonists reportedly enhanced GSIS through IP3-mediated Ca 2+ release from the ER-and diacylglycerol (DAG)-mediated activation of protein kinase C (PKC) (Yamada et al., 2016). Because the Ca 2+ concentration in the ER after stimulation with the GPR40 agonist was not decreased in UCP2-overexpressing INS1 cells, IP3-mediated Ca 2+ efflux from the ER was impaired by excessive UCP2. We also found that the Ca 2+ concentration in the ER was decreased after glucose stimulation by overexpression of UCP2 in INS1 cells. Because methylglyoxal has been shown to impair the activity of SERCA, a calcium pump in the ER, without any changes of protein levels of SERCA (Zizkova et al., 2018), increased methylglyoxal in bUCP2Tg islets might decrease ER Ca 2+ uptake through SERCA. In MIN6 mouse b-cell lines, Ca 2+ uptake through SERCA after high glucose stimulation was inhibited by oligomycin, an inhibitor of ATP synthase (Moore et al., 2011), thus the reduction in ATP production in bUCP2Tg islets might affect SERCA activity. Because UCP2 was also shown to act as a Ca 2+ uniporter in mitochondria (Koshenov et al., 2020;Trenker et al., 2007), the decrease in the ER Ca 2+ concentration after glucose stimulation in UCP2-overexpressing INS1 cells might be caused by increased Ca 2+ uptake through UCP2.
We also showed decreased expression of Calr in bUCP2Tg islets. Knockdown of calreticulin in MEFs reduced Ca 2+ storage in the ER and impaired IP3-dependent Ca 2+ release from the ER (Mesaeli et al., 1999). However, the basal Ca 2+ concentration in the ER was not changed by UCP2 overexpression in INS1 cells. Because increased calreticulin was reported to inhibit Ca 2+ release from the ER because of its high affinity for Ca 2+ in C. elegans (Burkewitz et al., 2020), the reduced Calr expression in bUCP2Tg islets might be the adaptive response against the impairment of Ca 2+ release from the ER. Taken together, our data suggest that the impairment of Ca 2+ release from the ER reduces GSIS in bUCP2Tg b-cells.
Given the role of UCP2 in Ca 2+ uptake by mitochondria in HeLa cells (Madreiter-Sokolowski et al., 2016), it is possible that impaired Ca 2+ influx in bUCP2Tg b-cells is, at least in part, due to an upregulation of mitochondrial Ca 2+ uptake. Whether UCP2 has a role as a classic uncoupling protein, such as UCP1, is controversial (Nicholls, 2016). A previous report showed that overexpression of UCP2 does not reduce the mitochondrial membrane potential in rat INS-1 b-cells, suggesting that UCP2 does not act as an uncoupler (Galetti et al., 2009). Indeed, proton leakage in bUCP2Tg islets was not changed compared with that in WT islets. UCP2 has a half-life of 30 min, whereas the half-life of UCP1 is 30 h (Rousset et al., 2007). This difference in half-life among UCP proteins also indicates distinct physiological functions of UCP2. A previous report characterized UCP2 as a metabolite transporter capable of reducing glucose oxidation in mitochondria (Vozza et al., 2014). Our data also demonstrated a reduction in ATP production and reduced mitochondrial respiration in bUCP2Tg islets. Because decreased GSIS, reduced mitochondrial respiration, and impairment of Ca 2+ flux by excessive UCP2 in b-cells were ameliorated by AldB knockdown, UCP2-induced AldB contributed to the b-cell dysfunction of bUCP2Tg mice. In particular, AldB-mediated intracellular Ca 2+ regulation by UCP2 in our study may provide clues to the novel pathologic mechanisms underlying b-cell failure.
In summary, we provide evidence that the upregulation of UCP2 in stressed b-cells under diabetes decreased insulin secretion due to mitochondrial dysfunction and impairment of Ca 2+ release from the ER by inducing AldB expression. Therefore, inhibition of UCP2 or AldB might be a promising therapeutic strategy to improve insulin secretion in patients with diabetes.

Limitations of the study
Our study demonstrates the molecular role of UCP2 and AldB as regulators of insulin secretion in b-cells.
We have demonstrated that UCP2 induces AldB expression and suppresses insulin release via dysfunction in mitochondria or Ca 2+ release from the ER. Although we showed the requirement of AldB for impaired ll OPEN ACCESS 16 iScience 25, 104603, July 15, 2022 iScience Article insulin secretion in UCP2-overexpressing b-cells in in vitro or ex vivo experiments, it was not evaluated in vivo. An inducible b-cell-specific AldB conditional knockout model will be a convincing tool to clarify this issue. This study highlights the expression and roles of UCP2 and AldB in human islets. To confirm the pathological or therapeutic significance of these two genes, a study using islets from donors with type 2 diabetes needs to be performed. Due to the unavailability and the extensive phenotypic diversity in type 2 diabetes islets, further studies will be needed.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: